Journal:

Article Title: Phospholipase D2 Is Localized to the Rims of the Golgi Apparatus in Mammalian Cells

doi: 10.1091/mbc.02-04-0059

Figure Lengend Snippet: PLD2 localization with different organelles in rat NRK cells. Cells were prepared for immunofluorescence microscopy and incubated with rabbit PLD2 antibody PLD2-27 (A, D, G, and J) and costained with monoclonal antibodies to the ER marker BiP (B); the transferrin receptor (E), a marker of early endosomes and the plasma membrane; and lgp120 (H), a late endosome/lysosomal marker. (K) Cells were stained with caveolin-1, a marker for caveoli; the arrow corresponds to caveolin-1 localized to the plasma membrane. Images were merged to determine overlap between PLD2 (red) and the respective marker proteins (green; C, F, I, and L). Areas of maximal overlap are yellow. (L) Inset, enlargement of the region of overlap between PLD2 and caveolin-1 in the perinuclear Golgi apparatus. The arrow indicates areas where PLD2 puncta (red) either overlap completely with caveolin-1 (green) or are directly adjacent. Images are from projected Z-series. Bars, 10 μm.

Article Snippet: Purified monoclonal anti-rat transferrin receptor antibodies were purchased from Cedarlane Laboratories (Hornby, Ontario, Canada).

Techniques: Immunofluorescence, Microscopy, Incubation, Marker, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: I427E mutation of αH in surface 2 of ILK-pKD impairs binding of GFP–ILK to GST–kindlin-2 F2PH. (A) GFP or GFP–ILK co-expressed with FLAG–α-parvin in CHO cells bound to glutathione bead-immobilized GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L357A (L/A) as a negative control detected by immunoblotting. One representative blot for each construct tested is shown. The lane labeled ‘3%’ indicates 3% of input lysate. Bead loading was visualized by Ponceau S staining. (B) Quantification of GFP or GFP–ILK binding to GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A (mean±s.e.m.; n=4). (C) Representative immunoblots for pulldown of GFP–ILK mutants co-expressed with FLAG–α-parvin in CHO cell lysates by GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A. The lane labeled ‘3%’ indicates 3% of input lysate. Bead loading was visualized by Ponceau S staining. (D) Quantification of binding of GFP–ILK and GFP–ILK mutants to GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A (mean±s.e.m.; n≥3); *P<0.005 (Student's t-test). Pulldown quantification graphs are shown as bar charts with individual data points plotted (dots).

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Mutagenesis, Binding Assay, Negative Control, Western Blot, Construct, Labeling, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: Additional residues on αH in the ILK-pKD are implicated in the interaction with kindlin-2. (A) Ribbon diagram of helix-αH and surrounding residues in the ILK-pKD–α-parvin-CH2 co-crystal structure (PDB ID: 3KMW) generated with Chimera software (Pettersen et al., 2004). Residues selected for mutation are labeled and shown as a ball-and-stick representation. Conservation coloring is indicated using the same color scale as shown in Fig. 1A. (B,C) Pulldown of GFP–ILK or GFP–ILK mutants by GST–kindlin-2 F2PH and GST–kindlin-2 F2PH L357A (L/A) from CHO cell lysate co-overexpressing FLAG–α-parvin assessed by representative immunoblots (B) and quantified (C); mean±s.e.m.; n≥3; *P<0.001 (Student's t-test). (D,E) Pulldown of GFP–ILK or GFP–ILK mutants from CHO cell lysate co-overexpressing FLAG–α-parvin using GST–kindlin-2 329-368 or GST–kindlin-2 329-368 L/A were assessed by representative immunoblots (D) and quantified (E); mean±s.e.m.; n≥3; *P≤0.0006. (F,G) Pulldown of GFP–ILK or GFP–ILK mutants from CHO cell lysate co-overexpressing FLAG–α-parvin using GST or GST–kindlin-2 were assessed in representative immunoblots (F) and quantified (G); mean±s.e.m.; n=4; *P≤0.0001 (Student's t-test). Pulldown quantification graphs are shown as bar charts with individual data points plotted (dots). GST- protein loading is indicated by Ponceau S staining.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Generated, Software, Mutagenesis, Labeling, Western Blot, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: R243G/R334G double mutation of GFP–ILK (GFP–ILK RR/GG) impairs binding of the ILK to α-parvin. (A) Ribbon diagram of selected regions in the ILK KD–α-parvin-CH2 complex co-crystal structure (PDB ID: 3KMW) surrounding I244, F245, and S246, generated with Chimera software (Pettersen et al., 2004). Residues selected for mutagenesis are labeled and shown as a ball-and-stick representation. Conservation coloring is indicated using the same color scale as shown in Fig. 1A. (B,C) Pulldown of GFP–ILK or GFP–ILK RR/GG from CHO cell lysates co-overexpressing FLAG–α-parvin using GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L357A (L/A) assessed by representative immunoblots (B) and quantified (C); mean±s.e.m.; n=3; *P≤0.0001 (Student's t-test). (D) Diagram of the GFP nanotrap experiment. GFP–ILK was purified from lysate of cells co-expressing GFP–ILK and FLAG-α-parvin. The amount of co-purifying FLAG–α-parvin was assessed by immunoblotting and the FLAG:GFP ratio for each construct was calculated. (E,F) GFP-nanotrap co-purification of GFP–ILK constructs co-expressed FLAG–α-parvin in CHO cells was assessed by immunoblot from one experiment (E) and quantified (F) as a raw FLAG:GFP ratio. Dots represent single data points for each construct tested. The lane labeled ‘2%’ indicates the 2% input of lysate. (G) The FLAG:GFP ratio for each GFP or GFP–ILK construct tested is expressed relative to the FLAG:GFP ratio of the GFP–ILK control within each experiment, which is set to 1. The dataset includes the experiment shown in E and F (mean±s.e.m.; n≥4); *P≤0.0015; ns, statistically not significant, P>0.05 (Student's t-test). (H,I) Pulldown of GFP–ILK K220M from CHO cell lysate co-overexpressing FLAG–α-parvin using GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A assessed by representative immunoblot (H) and quantified (I); mean±s.e.m.; n=3; *P≤0.0001. Pulldown and co-purification quantification graphs are shown as bar charts with individual data points plotted (dots). GST-protein loading control for pulldown experiments is indicated by Ponceau S staining.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Mutagenesis, Binding Assay, Generated, Software, Labeling, Western Blot, Purification, Expressing, Construct, Copurification, Control, Staining

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: GFP–ILK mutants that are impaired in kindlin-2 binding localize poorly to focal adhesions. CHO cells stably expressing mCherry-paxillin were transiently co-transfected with FLAG–α-parvin and either GFP alone, GFP–ILK or one of the GFP–ILK mutants. Six hours after replating on fibronectin-coated glass-bottom dishes, live cells were imaged by epifluorescence (EPI) and/or TIRF microscopy as indicated. Images in each channel were linearly and uniformly adjusted, and cropped for clarity. Scale bar: 20 µm.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Microscopy

Journal: Journal of Cell Science

Article Title: Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading

doi: 10.1242/jcs.221184

Figure Lengend Snippet: The ILK–kindlin-2 interaction is important for normal cell spreading in HeLa cells. (A) Immunoblotting of shScr and shILK HeLa cells to show protein levels of ILK, kindlin-2, α-parvin, PINCH1 and vinculin. (B) Bar graph showing residual protein levels in shILK cells calculated relative to those in shScr cells (mean±s.e.m.); individual data points are indicated (dots; n≥4). (C) Immunofluorescence staining of endogenous vinculin in fixed shScr or shILK HeLa cells spread on fibronectin-coated glass coverslips and acquired by epifluorescence microscopy. Scale bars: 20 µm. (D) CellProfiler quantification of GFP-positive cell areas pooled across three independent experiments shown as box and whiskers plots indicating 10th and 90th percentile range. n=155 shScr+GFP cells, 169 shILK+GFP cells, 154 shILK+GFP–ILK cells, 162 shILK+I427E cells, 143 shILK+K423D cells, 137 shILK+K426D cells, 125 shILK+I413D cells; *, significantly different from shScr+GFP calculated using one-way ANOVA and Tukey's correction for multiple comparisons (P≤0.01); ns, statistically not significant; P>0.05. (E) Immunoblotting of shScr and shK2 HeLa cells to show protein levels of kindlin-2, ILK, and β-tubulin. (F) Bar graph showing residual kindlin-2 or ILK protein levels in shK2 cells calculated relative to those in shScr cells (mean±s.e.m.); individual data points are indicated (dots; n≥3). (G) Immunofluorescence staining of endogenous vinculin in fixed shScr or shK2 HeLa cells spread on fibronectin-coated glass coverslips and acquired by epifluorescence microscopy. Scale bars: 20 µm. (H) CellProfiler quantification of GFP-positive cells areas pooled across three independent experiments; n=143 shScr+GFP cells, 121 shK2+GFP cells, 145 shK2+GFP-K2 cells, 130 shK2+GFP-K2 LA cells. Data are shown as box and whiskers plot, with whiskers indicating the 10th and 90th percentile range. *, significantly different from shScr+GFP calculated using one-way ANOVA and Tukey's correction for multiple comparisons (P≤0.015); ns, statistically not significant. Microscopy images were linearly and uniformly adjusted for clarity.

Article Snippet: Primary antibodies against GFP (Rockland, catalog #601-101-215; Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862; Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis, MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000), kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at 1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000), PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonyl reductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas) (used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank, catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugated secondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) were purchased from commercial sources.

Techniques: Western Blot, Immunofluorescence, Staining, Epifluorescence Microscopy, Microscopy